Cellulases specific activities

Irwin, D. C., Spezio, M., Walker, L P. and Wilson, D. B. (1993). Activity studies of eight purified cellulases specificity, synergism, and binding domain effects. Blotechnol Bioeng 42,1002-1013. 

Figures 1 and 2 show positive correlation, although of varying degrees of goodness of fit, between the specific activities of endo ucanase, exoglucanase, and -glucosidase activities with the cellulose composition and degradation in digester feed, respectively. These data indicate a direct relationship between the cellulose content in the feed and cellulase enzyme complex production by the digester consortia. In this system, as in many others studied, cellulose is an effective inducer of cellulase secretion (67,68),...

Figure 2. Comparisons of cellulose degradation of the MSW feedstock with specific cellulase enzyme activities in sludge from 7 CSTR digesters operated under different retention times and various conditions of nutrient limitation.

However, we have observed that values obtained with crude extracts were only qualitative. Often, they did not accurately estimate the quantities of the individual enzymes present. Inhibitors were typically present that caused the underestimation of certain enzymes ie.g., ligninases Table II) and that could potentially mask less dominant enzymes. Also, certain polysaccharidases e.g., hemicellulases) were often overestimated due to the action of non-specific or synergistic enzymes e.g., other hemicellulases or cellulases) (9,14), This artifact resulted in low apparent recovery of a given activity and only moderate increases in specific activity upon purification of the major corresponding enzyme present, in spite of the fact that SDS polyacrylamide gels indicated good recovery and substantial removal of contaminants (14),... 

Lignocellulosic biomass is a valuable and plentiful feedstock commodity and its high cellulose and hemicellulose content (about 80% of total) provides considerable potential for inexpensive sugars production. However, enzymatic deconstruction of these polysaccharides remains a costly prospect. Strides in cellulase cost reduction have been made, yet further improvements are needed to reach the goal of 0.10/gal of EtOH expected to enable this new industry. Strategies to reach this goal will combine reduction in the cost to produce the needed enzymes as well as efforts to increase enzyme efficiency (specific activity). As this work proceeds, the more easily attained achievements will be made first, and thus the overall difficulty increases with time. 

During the semibatch experiments, vacuum filtration was applied at 4 d and 8 d after the start of saccharification, to remove the sugar product as filtrate. In selected semibatch experiments, ultrafiltration was applied to the vacuum filtrate to recover soluble enzymes. In other semibatch experiments, the vacuum filter cake was washed extensively with deionized water to remove any enzymes not bound to the solids. After filtration, pretreated corn stover slurry and 7 mL of solution (the ultrafiltration filtrate, or citrate buffer when ultrafiltration was not used) were added to the residual solids and bound enzymes, to replace the volume removed as filtrate during the ultrafiltration step. The additional substrate promotes further saccharification by reusing the cellulase enzymes. To promote further saccharification in a final set of semibatch experiments, additional cellulase at a specific activity of 5 FPU/g of fresh cellulose was added along with the fresh corn stover after vacuum filtration. 

Cellulase activities of T. reesei broths are normally reported to lie between 400 and 600 FPU/g total protein.188 Our work to assess the specific activities of T. reesei cellulase preparations has led us to the direct comparison of commercial cellulase products, typically highly selected T. reesei mutants, and reconstituted, purified cellulase enzymes (Table 33.4). Although the range of specific activities found from this internally consistent study generally agrees with the literature, our estimation of the... 

TABLE 33.4 Specific Activities of Various Trichoderma reesei Cellulase Preparations... 

For the assay of cellulase activity it is desirable to use a method by which the number of cellulase units can be directly determined. A number of quantities are defined in terms of units of enzyme, such as the concentration of an enzyme, normally given as units/ml., the specific activity defined as units/mg. of protein, and the molecular activity, defined as units//unole of enzyme. The determination of units of activity does not present any difficulties for enzymes where the change of the substrate can be expressed in absolute terms. However, for enzymes whose activity is not measured in terms of a chemical reaction but in terms of some physical change, such as a decrease in viscosity of the substrate, complications arise and it has not been possible to express the activity in the units mentioned above. 

An insoluble, enzyme preparation (sedimenting at 100,000 g) has been obtained by Glaser it is capable of catalyzing the transfer of D-gluco-pyranosyl residues from uridine 5-(a-D-glucopyranosyl-C pyrophosphate) to form a radioactive, water-insoluble, alkali-insoluble polysaccharide. This product was identified as cellulose by (a) hydrolysis with the cellulase of Myrotkecium verrucaria, and (b) identification, as cellobiose, of the radioactive disaccharide obtained by acid hydrolysis—by repeated recrystallization with authentic cellobiose without loss of specific activity. 

Readers wishing to convert from cellulase loading in milligram protein per gram solids to filter paper units per gram solids may note that the specific activity of T. reesei cellulase preparations is about 0.6 filter paper units mg protein. 

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